Ateneo - UPO

Measuring biological things.
Techniques for sample preparation in biochemistry
Spectrophotometry.
Chromatography
Protein characterization.
Using antibodies in biotechnology.
Enzymes in diagnostic and research.
Binding assays.

"Introduzione alla biochimica di Lehninger"
David L. Nelson &Michael M. Cox
Zanichelli

Wet laboratory exercises aim at acquiring the practical skills required for the use of basic biochemical techniques and for a profitable thesis internship.
The laboratory theory lessons present advanced techniques that the students encounter in the scientific literature.

The students MUST have successfully attended the "safety in chemical and biological laboratories" course.
The knowledge on the structure and properties of molecules provided by organic chemistry are preparatory.

The course includes:
- lectures on the main techniques,
- simulations using specific software for the key techniques used in biochemistry laboratories,
- practical exercises in chemical-biological teaching labs.
Attendance is compulsory. In order to obtain the laboratory grade, each student must attend at least 75% of the classes (i.e., at least 22 out of the total 30 hours of the course).

At the end of the practical sessions, each student is required to submit a laboratory notebook, compiled during the activities, documenting the procedures carried out and the understanding of the contents. A similar submission is required at the end of the “virtual” exercises performed with simulation software.
On the last day of the practical laboratory, a verification test will be administered to assess the skills acquired and the understanding of the concepts covered in the lectures and exercises. The test will include multiple-choice questions, open-ended questions, and applied exercises.
The final grade will be based on the integration of the test results with the quality of the report and the laboratory notebook.

Students with physical disabilities, Learning Disabilities or Special Education Needs can request
specific services and tools via the
, consulting the University webpage: https://www.uniupo.it/en/services/services-students-physical-or-learning-disabilities

Students with disabilities, learning disabilities or special education needs, once they have contacted the University Staff, can refer to the tutor in charge of the course to define the examination
modalities, concerning academic aspects.

All students who have attended the theoretical lessons and the laboratory will be evaluated.
The final grade will be based on the integration of the test results with the quality of the report and the laboratory notebook.

Getting to know the laboratory and equipment: measuring, diluting, and preparing a calibration curve. Using Excel for constructing a calibration curve and practical exercises.
How to keep a laboratory notebook.
How to read and interpret a protocol.
Theory and practical exercises on solution preparation.

The significance of measurement in a biological context: specificity, accuracy, precision, and reproducibility.

Techniques for sample preparation for biochemical analysis (homogenization, detergent lysis). Solvent extraction and selective precipitation, their application to the fractionation of biological compounds. Theory of centrifugation and preparative centrifugation techniques for the fractionation of biological compounds. Use of centrifuges and centrifugal force.

Principles of spectrophotometry. The theoretical foundations and practical aspects of spectroscopic techniques (absorption, fluorescence, polarimetry) and examples of their use for the quantification of biological analytes. Characteristics and use of a spectrophotometer and fluorimeter.

Principles of chromatography, main chromatographic techniques (affinity, gel filtration, ion exchange, and reverse phase) and related matrices. Use of chromatography for separating complex mixtures of proteins and lipids based on their chemical and physical properties. Equipment used (column chromatography, thin layer chromatography, HPLC, etc.). Detectors and results analysis (retention coefficients, qualitative and quantitative analysis).
Principles of electrophoresis with a focus on protein fractionation and characterization. IEF, SDS-PAGE, 2D-PAGE, capillary electrophoresis. Common detection systems: dyes and western blotting.

Use of enzymes in diagnostics and research. Characterization of enzyme kinetics and study of inhibitors. Use of enzymes in clinical diagnostics, kinetic and end-point assays applied to research and diagnostics. Analysis of an enzyme's kinetic parameters.

Binding and association assays, analysis of receptor-ligand equilibrium."

The students will acquire knowledge of the main biochemical methodologies, with particular emphasis on the study of proteins. This knowledge is useful for critically reading technical literature. The exercises enable effective navigation in the laboratory, also in preparation for the graduation internship. For this purpose, practice in maintaining the laboratory notebook is important.